TY - JOUR
T1 - Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems
AU - Chen, Shao Yung
AU - Dong, Mingming
AU - Yang, Ganglong
AU - Zhou, Yangying
AU - Clark, David J.
AU - Lih, T. Mamie
AU - Schnaubelt, Michael
AU - Liu, Zichen
AU - Zhang, Hui
N1 - Funding Information:
This work was supported in part by grants from the National Institutes of Health, National Cancer Institute, the Early Detection Research Network (EDRN, Grant U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC, Grant U24CA210985), and the National Institute of Innovation in Manufacturing Biopharmaceuticals (NIIMBL, Grant 0000049472) Project.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2020/1/21
Y1 - 2020/1/21
N2 - Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.
AB - Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.
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U2 - 10.1021/acs.analchem.9b03761
DO - 10.1021/acs.analchem.9b03761
M3 - Article
C2 - 31859482
AN - SCOPUS:85078396741
VL - 92
SP - 1680
EP - 1686
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 2
ER -